Abstract:
The renaturation efficiency of recombinant prochymosin depends on not only the renaturation conditions but also the solubilization (denaturation) conditions. Compared with pH 8, solubilization of prochymosin-containing inclusion bodies at pH 11 (8 mol/L urea) results in onefold increase of renaturation efficiency (～40% vs. ～ 20 %). Alkaline pH facilitates the solubilization of inclusion bodies via the breakage of intermolecular disulfide bonds. Moreover, alkaline pH renders prochymosin molecules to be in a more reduced and more unfolded state which undergoes refolding readily.

Abstract:
The design of any antagonist or inhibitor for any enzyme requires the knowledge of structure-
function relationship of the protein and the optimum conformational states for maximum and minimum activities. Furthermore, designing of the inhibitors or drugs against an enzyme becomes easier if there is information available about various well characterized intermediate conformation of the molecule. In vivo folding pathway of any recombinant protein is an important parameter for understanding its ability to fold by itself inside the cell, which always dictates the downstream processing for the purification. In the present manuscript we have discussed about the in vivo and in vitro folding, and structure-function relationship of Dihydrofolate reductase enzyme. This is an important enzyme involved in the cell growth and hence inhibition or inactivation of the enzyme may reduce the cell growth. It was observed that the equilibrium unfolding transition of DHFR proceeds through the formation of intermediates having higher exposed surface hydrophobicity, unchanged enzymatic activity and minimum changes in the secondary structural elements. Because of enhanced surface hydrophobicity, and unchanged enzymatic activity, these intermediates could be a nice target for designing drugs against DHFR.

Abstract:
five different detergents for restoration of igg antibody binding were studied in the washing steps and in the buffer solution used for the dilution of the conjugate and samples. binding of human serum igg antibodies to outer membrane protein (omp) was detected with class-specific peroxidase-conjugated human antibodies. the positive control reacted with those proteins whose molecular weight corresponded approximately to p1, p3, p4 and p5. proteins of 80 kda, 70 kda and 24 kda and another protein of >150 kda were also recognized. our studies indicated that tween 20 was the best detergent for restoration of omp antigenicity, except for the 150 kda protein. tween 20 achieved a greater number and intensity of bands and a lower background with respect to empigen bb, triton x-100, nonidet np-40 and chaps. the use of detergents affected the antigenicity of the 150 kda protein. washing with tween 20 were the most important steps for restoration of igg antibody binding sites of heat-denatured omps.

Abstract:
Large scale abstraction and isolation of bacterially synthesized, recombinant-DNA-derived, porcine growth hormone (r-pST) is described. The r-pGH is found in genetic engineering E.coli as the form of inclusion bodies. Pellet fraction which were mainly inclusion bodies, after cell breakage and centrifugation, were collected. Cell envelope components, such as protein, lipid, endotoxin and nucleic acids are selectively removed from the pellet fraction by an EDTA/lysozyme/deoxycholate extraction. Inclusion bodies were dissolved using 6mol/L guanidine/HCl and air oxidation is then carried out in the presence of the guanidine/HCl. The Guanidine/HCl protein mixture were diluted by renaturation solution. Guanidine/HCl were removed by dialysis and then correctly refolded, oxidized r-pGH were obtained. Injection experiment of hypophysectomized rats proved r-pST with high native bioactivity was obtained.

A detailed molecular model for alkali-denatured duplex
circular DNA (“Form IV”) is proposed. The illustrative biological example used is
the replicative form of fx174, a 5 kb duplex
circular chromosome. The model explains all of Form IV’s known and peculiar features.
In a sedimentation coefficient vs. pH titration, Form IV begins to appear at pH
12.3, at which point it can be persuasively argued that no further supertwists can
be added to the already-highly-supertwisted chromosome. Therefore a new structure
must appear. The sedimentation coefficient s then undergoes a massive, but
initially reversible increase as the pH is raised further, culminating at pH 12.8
with a 250% increase. This degree of compactness can only be explained by a 4-stranded
tetraplex structure, consisting of a pair of duplexes whose base pairs are mutually
intercalated. Above pH 12.8, the structural changes become irreversible, suggesting a further conformational
change. It is proposed that this involves an axial rotation of the component duplex
strands, so that the bases now stack on the outside, and the phosphate groups lie
in the core, where they bond ionically by means of salt bridges. When the irreversibly
denatured compact structure is neutralized at moderate-to-high salt concentrations,
a third novel structure appears, which has a sedimentation coefficient midway between
the native 21 s and the
denatured 50 s. It is proposed that this is a hybrid structure; part tetraplex,
part duplex. To return to a fully-duplex form, it is necessary to both neutralize
the solution, and also to greatly reduce the ionic strength, i.e., to the range 0.001-0.01 M. Since the DNA, under those conditions,
cannot possibly have normal complementary base-pairing, the duplex structure must
either be tautomerically base-paired, or else stabilized solely by base-stacking,
with no base-pairing at all.

Abstract:
Recombinant human pro-urokinase forms insoluble inclusion body when overexpressed in Escherichia coli, and it must be denatured and renatured in vitro before it acquires activity. This study aimed to increase the renaturation yield of denatured pro-urokinase. We have evaluated the basic renaturation conditions of pro-urokinase through qualitative and quantitative analysis of pH, temperature, denaturant concentration, protein concentration, the ratio of reduced and oxidized thiol reagents. The effects of nonspecific additives, step-wise dilution and urea gradient dialysis have been also compared. The optimal conditions of pro-urokinase renaturation with the yield about 20%-30% have been obtained.

Abstract:
Most of single chain Fv fragment (ScFv) of monoclonal antibody deposited as insoluble inclusion bodies during expression in E. coli. In order to convert inactive and misfolded inclusion body ScFv into soluble products, the renaturation and purification procedure was developed, recombinant ScFv was redissolved, diluted, and was directly loaded onto a Sephadex G-25 column. The size-exclusion chromatography provided a favorable environment for the proteins to renature, it also spatially constrained partially refolded ScFv from aggregating and diffusing toward each other, promoted renaturation. In the size-exclusion chromatography, the ScFv protein flowed faster and was eluted earlier than the denaturant, this will allow the protein sample to pass through decreasing denaturant concentrations, which promotes ScFv refolding and removes denarurant. During this process, ScFv was simultaneously purified, and at least 150mg/L (with the purity more than 95 %) soluble ScFv was obtained. The strategy provides an economic and novel approach for the production of ScFv from inclusion body proteins.

Abstract:
Recombinant proteins and enzymes are commonly used in many areas of our life, such as diagnostics, industry and medicine, due to heterologous synthesis in prokaryotic expression systems. However, a high expression level of foreign protein in bacteria cells results in formation of inactive and insoluble aggregates – inclusion bodies.Reactivation of aggregated proteins is a complex and time-consuming process. Every protein requires experimental optimization of the process conditions. The choice of the refolding method depends on the type of recombinant protein and its physical, chemical and biological properties.Recovery of the activity of proteins accumulated in inclusion bodies can be divided into 4 steps: 1) inclusion bodies isolation, 2) solubilization of aggregates, 3) renaturation, 4) purification of catalytically active molecules.Efficiency of the refolding process depends on many physical factors and chemical and biological agents. The above parameters determine the time of the folding and prevent protein aggregation. They also assist the folding and have an influence on the solubility and stability of native molecules.To date, dilution, dialysis and chromatography are the most often used methods for protein refolding.

Abstract:
A recombinant RGD-Staphylokinase(RGD-Sak) with thrombolytic and anti-thrombolytic bifunction was expressed in E.coli. The expression product accumulates as inclusion bodies. In order to obtain active molecule, the RGD-Sak in the inclusion body should be denatured and then renatured. The renaturation of RGD-Sak was performed by gel filtration. Comparing with the traditional way of dilution renaturation, gel filtration way is better than the traditional one, since there are some advantages, such as simple processing, high recovery, low cost and higher purity after renaturation. After renaturation, RGD-Sak was purified by Q-Sepharose FF, and the purity was more than 95%. Analysis of CD spectra showed that the final product from the two renaturation ways have similar CD spectra. It was demostrated that RGD-Sak molecules proceeded correct refolding through gel filtration or dilution renaturation process.